DESCRIPTION: ( Investigator's abstract) In hereditary spherocytosis (HS), defects of spectrin ankyrin, band 3 and protein 4.2 lead to spheroidal, osmotically fragile cells that are selectively trapped in the spleen. The prevailing theory is that HS is caused by local disconnection of the skeleton and bilayer, followed by vesiculation of the unsupported surface components. The mechanism of this effect is not well understood. One possibility is that the lipid bilayer is stabilized directly by interactions with the membrane skeleton. Alternatively, the lipids may interact with the multiple transmembrane helices of band 3, which would indirectly anchor them to the skeleton. These two hypotheses will be tested using the technique of targeted gene replacement in mouse embryonal stem (ES) cells. Specifically, band 3 will be replaced with: (1) a band 3 derivative that lacks monovalent anion transport function, (2) a chimera of the glucose transporter attached to the cytoplasmic domain of band 3, (3) the isolated membrane domain of band 3, and (4) the isolated cytoplasmic domain of band 3 anchored to the membrane by a single transmembrane helix. The effects of each mutation on survival, organ function and pathology, red cell lifespan and morphology, red cell membrane and membrane skeletal composition and organization, membrane stability, and the function and mobility of band 3 will be investigated. The spectrin-based membrane skeleton is widely distributed in nature, but relatively little has been done to investigate its functions outside of the red cell, where a great deal is known. Recent work indicates that some spectrins (and other membrane proteins) attach preferentially to internal vesicles and organelles. We have recently isolated two new spectrins that associate with intracellular vesicles or organelles. Spectrin beta IV is particularly interesting because it is the first truncated spectrin and because one of its major isoforms may have the ability to polymerize end-to-end to form linear polymers. We will clone and characterize these isoforms, determine where they are located in cells and tissues, identify their binding partners, develop assays to measure their binding and polymerization in vitro, and determine the consequences of overexpressing specific isoforms, expressing dominant negative forms and knocking out the beta IV spectrin gene. We expect this interesting spectrin will prove to have important intracellular responsibilities and will help expand knowledge of membrane skeletons from the periphery to the interior of the cell.